KR101746667B1 - Chimeric peptide having antialgae activity and composition for control of red tide comprising the same - Google Patents
Chimeric peptide having antialgae activity and composition for control of red tide comprising the same Download PDFInfo
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- KR101746667B1 KR101746667B1 KR1020150142216A KR20150142216A KR101746667B1 KR 101746667 B1 KR101746667 B1 KR 101746667B1 KR 1020150142216 A KR1020150142216 A KR 1020150142216A KR 20150142216 A KR20150142216 A KR 20150142216A KR 101746667 B1 KR101746667 B1 KR 101746667B1
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- KR
- South Korea
- Prior art keywords
- peptide
- seq
- red tide
- amino acid
- dwr
- Prior art date
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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Abstract
The present invention relates to a chimeric peptide having an anti-algae activity, and a composition for controlling red tide containing the same as an active ingredient, which comprises Hn-Mc peptide consisting of the amino acid sequence of SEQ ID NO: 1, Hn-Mc -Del peptide, the Hn-Mc-DRW peptide consisting of the amino acid sequence of SEQ ID NO: 3, or the Hn-Mc-DWR peptide consisting of the amino acid sequence of SEQ ID NO: 4 inhibits the motility and growth of the blooming microalgae, It is possible to inhibit photosynthesis by destroying the membranes of cell membranes, intracellular organelles, or photosynthesis by binding to the cell membranes of the micro tide which does not contain red tides. Furthermore, since it does not show cytotoxicity against fish and shellfishes, May be useful as a composition.
Description
The present invention relates to a chimeric peptide having an anti-algae activity and a composition for controlling red tide containing the chimeric peptide as an active ingredient. More specifically, the present invention relates to a chimeric peptide having anti- A chimeric peptide which inhibits the movement and growth of microalgae and is not toxic to fish and shellfish, and a composition for controlling red tide containing the chimeric peptide as an active ingredient.
Red tide refers to a phenomenon in which a living creature (eg, plankton) that meets the appropriate environment to reproduce and reproduces in large quantities changes the color of the seawater to red or green / brown.
The damage caused by these red tides can be divided into two major categories. Firstly, there are massive deaths and deaths of fish and shellfishes, and another is the damage of tourism industry. Specifically, fish and shellfish eat the living creatures in the process of sucking the water to catch the food, and the fish and shellfish are killed by the toxins of the living creatures. In the case of large-scale propagation of suspended organisms, nutrients such as nitrogen and phosphorus are insufficient, and suspended organisms are temporarily drowned and settled on the seabed. In order to decompose them, the bacteria consume a large amount of oxygen dissolved in seawater, Fish and shellfish suffocate due to lack of oxygen and die. In addition, red tides often occur during late spring and summer, when the temperature is high and windy, and this is the time when many tourists visit the beach. When tourists swim, they can drink water containing toxic red tide creatures and die or become paralyzed. have. Many European countries, including the United States, Spain, and Italy, have closed their beaches during this period and have suffered tremendous damage to tourism income. In Korea, red tides occur in the wide sea area of the southern coast and occur in the east coast and west coast of the southern coast. Especially, in the case of west coast, red tide is repeated every year in Chunsu Bay from 1993 and Jeonbuk coast from 1995. Since 1998, harmful red tide has occurred on a large scale.
There are more than 200 kinds of creatures that cause red tide, and 40 kinds of red tide creatures appear in the coast of Korea. Specifically, Cochlodinium polykrikoides sp. Was first found on the coast of Puerto Rico and then bloomed in Bamagat Bay, New Jersey, on the Atlantic coast of North America, and on the coast of California, USA It is regarded as a species such as Cochlodinium heterolobatum (Margalef, 1961; Silva, 1967). This species has been reported to have caused red tides in central and western Japan since it caused fisheries damage by causing red tides in Harima Nada and Yatsushiro Bay in Japan (Yuki and Yoshimatsu, 1989; Fukuyo et al., 1990). Cochlodinium polykrikoides sp. Have recently been reported to cause red tides also in the Pacific coast of Guatemala, in the Gulf of California, Canada, and in Latin America (see Whyte et < RTI ID = 0.0 > al. , 2000; Carate Lizarraga et al. , 2000). Prorocentrum minimum , Prorocentrum micans and Heterosigma akashowi , Chattonella sp., And Chateaunella sp., Belonging to the spiny molluscs, Chattleella marina is also known to cause red tides. In the case of Chattonella sp., When the density of red tide is more than 50, it occurs over a radius of 2 ~ 5 km, and a warning of red tide is issued due to concern about fishery damage. When the density of suspended creatures is 100 or more, And significant fishing damage is anticipated, and a red alert is issued.
Accordingly, various methods have been studied in the art to prevent red tide or to reduce damage caused by red tide. Specifically, chemical and physical methods have been developed and used to remove red tide organisms by spraying chemicals, using an ultrasonic pulverizer, an ozone generator, and a centrifuge, or by adsorbing red tide-causing organisms by using clay to deposit on the floor. However, these methods may have another adverse effect on the ecosystem, and there are many problems to be used in a wide range of red tide generation areas. To solve this problem, biological methods for eliminating unwanted organisms by using prey-predator relationships in ecosystems have been researched and developed, but this method can cause another problem by changing the food chain of the ecosystem. In addition, methods of killing the organisms causing red tide by using substances secreted by marine bacteria have been actively studied, and recently, antibiotics acting on protein synthesis inhibitors, cell wall inhibitors and cell membranes have been used, Attempts have been made to artificially control the red tide induced organisms by organic synthesis of low molecular substances. However, such antibiotics can accumulate in the ocean through ecosystems or food produced in the sea, such as the problems caused by pesticides used in agriculture, which are not decomposed at sea.
Considering these problems, peptides and protein preparations can be used as anti-redeposition agents that can decompose naturally and are harmless to the human body and natural ecosystem. Not only can they be mass-expressed through genetic manipulation to reduce production costs, but they can also produce peptides on the surface of bacteria or viruses that specifically adhere to red tide organisms, resulting in anti-red cell activity.
In this regard, Mastoparan B, an antimicrobial peptide having an amphipathic alpha helical structure purified from bee venom, was shown to be effective against algae Alexandrium tamarense and Chattonella catenatum , Has been confirmed. However, this peptide has been reported to exhibit high cytotoxicity at low concentrations.
Accordingly, the inventors of the present invention have conducted studies on chimeric peptides showing low cytotoxicity and excellent anti-alveolar activity. While using 15 to 17 amino acid residues in general, it is possible to produce economically large quantities, (Hn-Mc-Del, Hn-Mc-DRW, or Hn-Mc-DWR), which inhibits growth and is not toxic to fish and shellfishes. The chimeric peptide And inhibits or kills the growth of microalgae causing red tides. As a result, it has been confirmed that the composition can be used as an excellent red tide control composition, and the present invention has been completed.
It is an object of the present invention to provide a chimeric peptide having no cytotoxicity and showing excellent anti-alveolar activity against red tide-induced microalgae.
It is another object of the present invention to provide a composition for controlling red tide containing the chimeric peptide as an active ingredient.
In one embodiment, the present invention provides a chimeric peptide comprising an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4 and exhibiting an anti-alveolar activity.
The term " algae " used in the present invention means inhibiting the growth of microalgae, such as inhibiting the growth or motility of microalgae causing red tide phenomenon, and killing microalgae.
In the present invention, the chimeric peptide consisting of the amino acid sequence of SEQ ID NO: 1 was designed by the present inventors and patented (Application No. 10-2015-0032977). The amino terminal region (1-7) of the HPA3NT3 peptide And a carboxyl terminal region (17-26) of melittin are conjugated to each other.
In the present invention, the chimeric peptide comprising the amino acid sequence of SEQ ID NO: 1 is referred to as "Hn-Mc peptide ".
In the present invention, the chimeric peptide comprising the amino acid sequence of SEQ ID NO: 2 is obtained by deleting the 16th and 17th glutamine (Q) of the Hn-Mc peptide.
In the present invention, the chimeric peptide comprising the amino acid sequence of SEQ ID NO: 2 is referred to as "Hn-Mc-Del peptide ".
In the present invention, the chimeric peptide comprising the amino acid sequence of SEQ ID NO: 3 comprises serine (S), which is the 9th amino acid of the Hn-Mc-Del peptide, as arginine (R) arginine, R) with tryptophan (W).
In the present invention, the chimeric peptide comprising the amino acid sequence of SEQ ID NO: 3 is referred to as "Hn-Mc-DRW peptide ".
In the present invention, the chimeric peptide consisting of the amino acid sequence of SEQ ID NO: 4 comprises a first amino acid of phenylalanine (F) as a tryptophan (W) and a 9th amino acid serine serine, S) with arginine (R).
In the present invention, the chimeric peptide comprising the amino acid sequence of SEQ ID NO: 4 is referred to as "Hn-Mc-DWR peptide ".
According to one specific embodiment, the chimeric peptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 of the present invention is useful for the growth of blooming microalgae And mobility.
Particularly, the chimeric peptide comprising the amino acid sequence of SEQ ID NO: 4 of the present invention has a bacterium inducing effect even when the concentration is 4 to 16 times lower than the chimeric peptide having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: It was confirmed that it inhibits the movement and growth of microalgae and binds to the cell membrane of red tide induced microalgae without cellulosic cell wall to destroy membrane of cell membrane and intracellular organelles or to inhibit photosynthesis of red tide induced microalgae to induce the death of microalgae .
At this time, the red tide caused microalgae Alexandria Solarium Kata Nella (Alexandrium catanella), Alexandria Solarium Tama lances (Alexandrium tamarense), Chateau Nella Marina (Chattonella marina), Chateau Nella SP (Chattonellar sp.), Kokeulrodinium poly Cri Koh des (Cochlodinium polykrikoides), Jiro di nium impyu dikeom (Gyrodinium impudicum), heteroaryl Sigma Akashi Wu (Hetrosigma akashiwo), heteroaryl Sigma sP (Heterosigma sp.) pro cent ROM mikan's (Prorosentrum micans) and professionals in cents ROM minimum (Prorocentrum minimum) , But it is not limited to these microalgae.
Therefore, the chimeric peptide comprising the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4 of the present invention can be usefully used as a composition for controlling red tide.
In another aspect, the present invention provides a composition for controlling red tide comprising a chimeric peptide comprising an amino acid sequence of any one of SEQ ID NOS: 1 to 4 as an active ingredient.
The anti-redeposition composition of the present invention may further contain, as an active ingredient, a chimeric peptide consisting of an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4 having an anti-alveolar activity and an optional component exhibiting anti-alveolar activity .
The composition for controlling anti-redness of the present invention may be prepared in various forms according to known methods, and a chimeric peptide consisting of the amino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 4 may be mixed with water or an organic solvent , Stability of effect and adhesion of the drug to the target organism may be used together with non-ionic or ionic surfactant. Further, it may further include an adsorbent, a co-agent, a dispersant, a binder, a flow aid, and the like.
The composition for controlling red tide of the present invention can be treated in seawater by a conventional method to control red tide induced microalgae. For example, the red tide controlling composition of the present invention can be sprayed once or several times to the ocean at an appropriate concentration. The number of doses, intervals of administration, and the concentration of administration can be controlled according to the degree of proliferation of the microalgae induced by blooms and the environmental conditions such as climate.
An Hn-Mc-DR peptide consisting of the amino acid sequence of SEQ ID NO: 1, an Hn-Mc-Del peptide consisting of the amino acid sequence of SEQ ID NO: 2, an Hn-Mc-DRW peptide consisting of the amino acid sequence of SEQ ID NO: 3, Hn-Mc-DWR peptide consisting of the amino acid sequence of bovine molluscs inhibits the motility and growth of red tide-induced microalgae and binds to the cell membrane of red tide-induced microalgae without cellulosic cell wall to destroy membranes of cell membranes and intracellular organelles or inhibit photosynthesis Induced microalgae and does not show cytotoxicity against fish and shellfish, it can be usefully used as a composition for preventing red tide in a marine ecosystem.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a photomicrograph showing the anti- alveolar activity against C. polykrikoides , a haploid algae of the Hn-Mc-DWR peptide of the present invention;
FIG. 2A is a graph showing the erythrocyte hemolytic activity of the Hn-Mc peptide, the Hn-Mc-Del peptide, the Hn-Mc-DRW peptide, or the Hn-Mc-DWR peptide of the present invention in the blood of red sea bream.
FIG. 2B is a graph showing the cytotoxicity of the Hn-Mc peptide, Hn-Mc-Del peptide, Hn-Mc-DRW peptide or Hn-Mc-DWR peptide of the present invention to fish cells (CHSE-214) .
A of FIG. 3 is a graph showing the cell membrane destruction function for Centrum minimum (P. minimum) as hetero Sigma Akashi Wu (H. akashiwo) and pro-Mc-Hn DWR peptides of the invention.
FIG. 3B shows the change in the content of H-akashwo and chlorophyll a in P. minima by Hn-Mc-DWR peptide of the present invention with time Graph.
FIG. 4 is a graph showing the effect of the anti-hu-Mc-DWR peptide of the present invention on the anti-scallop shell of H. akashiwo , P. minimum and C. marina treated with the anti- It is a photograph showing the bird activity.
Hereinafter, the present invention will be described in more detail by way of examples and the like. It will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples and the like according to the gist of the present invention. will be.
Example 1 Preparation and Separation of Peptides Having Antifungal Activity
The HPA3NT3 (1-7) portion, which is amphiphilic and cationic in the amino terminal region of HPA3NT3, which is a derivative of Helicobacter pylori secretion peptide HP (2-20), and the amphiphilic and cationic portion of the carboxyl terminal region of melittin Hn-Mc peptide having an amphipathic α-helix structure with increased hydrophilicity and cationicity and having the amino acid sequence of SEQ ID NO: 1 was prepared by conjugating melittin (17-26) having the amino acid sequence of SEQ ID NO: 1.
Also, Hn-Mc-Del peptide having the amino acid sequence of SEQ ID NO: 2 was prepared by deleting the 16th and 17th glutamine of the Hn-Mc peptide.
The 9th serine of Hn-Mc-Del was substituted with arginine, the 15th arginine was replaced with tryptophan, and the Hn-Mc-DRW peptide having the amino acid sequence of SEQ ID NO: .
The first phenylalanine of the Hn-Mc-Del was substituted with tryptophan and the 9th serine was substituted with arginine to obtain the Hn-Mc-DWR peptide consisting of the amino acid sequence of SEQ ID NO: .
The peptides were synthesized using Merrifield's liquid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as an amino protecting group. The peptides in which the carboxyl terminal is in the -NH 2 form in the above peptide were used as the starting material of Rink Amide MBHA-Resin, and a peptide chain of Fmoc-amino acid by coupling of Fmoc- The extension was carried out by N-hydroxybenzotriazole-dicyclo-hexycarbodiimide (HOBt-DCC).
Specifically, the Fmoc-amino acid of the amino terminal of each peptide was coupled and then the Fmoc group was removed with 20% piperidine / dimethyl formamide (hereinafter referred to as 'DMF') solution, and DMP and dichloromethane washed several times with dichoromethane (DCM), and then dried with nitrogen gas. Trifluoroacetic acid (TFA): phenol: thioanisole: H 2 O: triisopropylsilane = 85: 5: 5: 2.5: 2.5 (vol: trifluoroacetic acid: phenol: thioanisole: water: ./vol.)] solution was added and reacted for 4 hours to remove the protecting group. Then, the peptide was separated from the resin and reacted with cold diethyl ether for 30 minutes. The reaction product was then centrifuged to precipitate the peptide, and the peptide precipitated with diethyl ether was washed once more, precipitated and dried in the air. The crude form of the peptide thus obtained was dissolved in ammonium bicarbonate at pH 8, and purified on a reverse phase-HPLC column (Delta Pak, St. Louis, MO) using acetonitrile as a
As shown in Table 1, all of the peptides synthesized according to the present invention exhibited a purity of 95% or more, and the measured molecular weights corresponded to the molecular weights calculated from the amino acid sequences. Hn-Mc peptide having the correct amino acid sequence 1), Hn-Mc-Del peptide (SEQ ID NO: 2), Hn-Mc-DRW peptide (SEQ ID NO: 3) and Hn-Mc-DWR peptide (SEQ ID NO: 4) were synthesized.
<Experimental Example 1> Measurement of anti-alveolar activity of peptides
≪ 1-1 > Measurement of 90% motility and killing concentration of bivalent microalgae of the peptide according to the present invention
The anti-alveolar activity of the Hn-Mc peptide, Hn-Mc-Del peptide, Hn-Mc-DRW peptide, or Hn-Mc-DWR peptide synthesized in Example 1 was measured. Specifically, red tide-inducing microalgae for measuring antifungal activity include Alexandrium catanella , Alexandrium tamarense , Chattonella marina , Chattonellar sp. But are not limited to, Cochlodinium polykrikoides , Gyrodinium impudicum , Hetrosigma akashiwo , Heterosigma sp . Prorosentrum micans , The Prorocentrum minimum was sold from the Korea Marine Microalgae Bank. Each of the microalgae thus dispensed was diluted with F2 medium (150 mg NaNO 3 , 8.69 mg NaH 2 PO 4 .9H 2 O, 10 mg ferric EDTA, 0.22 mg MnCl 2 , 0.11 mg CoCl 2 , 0.0196 mg CuSO 4 .5H 2 O, ZnSO 4揃 7H 2 O, 50 mg Na 2 SiO 3揃 9H 2 O, 0.012 mg Na 2 MoO 4揃 2H 2 O, 1 Vit Vitamine B 12 , 1 Bi Biotin, 0.2 쨉 g Thiamine HCl / The cells were incubated at a temperature of 22 캜, a salinity of 34 ‰, and an illuminance of 3,000 lux at a constant light intensity cycle of 16 hours / 8 hours. Thereafter, the cells were diluted so that the number of cells of the cultured microalgae was 10 4 / ml and inoculated into a 24-well microtitre plate. The Hn-Mc peptide, Hn-Mc-Del peptide , Hn-Mc-DRW peptide, or Hn-Mc-DWR peptide was diluted 1/2 times starting from the 64 mg / L concentration and added to the plate to which the strain had been inoculated. Thereafter, the number of red tide organisms was measured using a hemocytometer every 2 hours while culturing for 36 hours under the culture conditions mentioned above. 90% of the immotile concentration (90% of the immotile concentration, hereinafter abbreviated as 'IC 90 ') and 90% of the algal concentration (90% of the algicidal concentration, abbreviated as AC 90 ) Respectively. The results are shown in Table 2.
As shown in Table 2, Hn-Mc-Del peptide, Hn-Mc-DRW peptide or Hn-Mc-DWR peptide had a concentration of inhibiting 90% motility of all blooming microalgae of 8 mg / mL, and the 90% kill concentration was less than 32 mg / mL. Especially, Hn-Mc-DWR peptide showed the lowest inhibition of mobility and growth of blooming microalgae.
Therefore, it was found that the Hn-Mc peptide, Hn-Mc-Del peptide, Hn-Mc-DRW peptide, and Hn-Mc-DWR peptide prepared in Example 1 of the present invention all had anti-alveolar activity.
<1-2> Anti-algae activity of Hn-Mc-DWR peptides in the red tide-induced microalgae without cellulosic cell walls
To visualize the anti-algae activity of the Hn-Mc-DWR peptide synthesized in Example 1 on an optical microscope, C. polykrikoides , which is a parasitoid alga, was inoculated on F2 medium, -1. ≪ / RTI > Cultured C. polykrikoides was inoculated into a 24 well microtitrate plate at a concentration of 10 4 / ml, and Hn-Mc-DWR peptide was further added at a concentration of 2 mg / L, And cultured for 24 hours. After incubation, they were observed under an optical microscope for visualization by incubation time. The results are shown in Fig.
As shown in Fig. 1, it was confirmed that the number of organisms was maintained and no external change was observed in the case of no treatment of C. polykrikoides as a red tide-inducing microalgae (Fig. 1A). On the other hand, after 1 min of treatment with Hn-Mc-DWR peptide at a concentration of 2 mg / L, the biomass of C. polykrikoides was observed to break up the chain and burst the
Thus, the anti-alveolar activity of the Hn-Mc-DWR peptide of the present invention was confirmed.
<Test Example 2> Cytotoxicity test of peptide according to the present invention
In order to confirm whether the anti-algae peptides of the present invention exhibit cytotoxicity, the present inventors investigated the erythrocyte destruction and cytotoxicity of anti-algae peptides.
Specifically, the red blood cells were washed 3 times with phosphate buffer solution (PBS, pH 7.0) so as to have a concentration of 8% in the blood of the red sea bream, and then diluted. Then, the red blood cells were continuously diluted with 1/2 mg from the concentration of 128 mg / The Hn-Mc peptide, the Hn-Mc-Del peptide, the Hn-Mc-DRW peptide or the Hn-Mc-DWR peptide synthesized in Example 1 were added and reacted at 37 ° C for 1 hour. Thereafter, the reaction solution was centrifuged at 1,000 x g and the amount of hemoglobin contained in the supernatant was measured at a wavelength of 414 nm. As a positive control, melittin, which has red cell destructive ability, was used.
To investigate the degree of cell destruction, the absorbance was measured by adding 1% Triton X-100, and the cell-destructive activity of 1% Triton X-100 was determined as 100% Were calculated according to the following equation (1).
Cytotoxicity was assessed by the addition of 10% Fetal bovine serum to chick salmon embryo cells (CHSE-214), a fish cell, in Eagle's minimum essential medium (MEM) Hn-Mc-Del peptide, Hn-Mc-DRW peptide or Hn-Mc-DWR peptide synthesized in Example 1 at the same concentration as the erythrocyte destructive capacity test and a positive control group treated with the melittin and after 24 hours, was treated XTT [(2,3-Bis- (2 -Methoxy-4-Nitro-5-Sulfophenyl) -2 H -Tetrazolium-5-Carboxanilide)] reagent And the degree of growth was confirmed. The results are shown in Fig.
(Absorbance A is the absorbance at 414 nm wavelength, absorbance B is the absorbance at 414 nm wavelength, and absorbance C is the absorbance at 414 nm wavelength and 1% Triton X- 100). ≪ tb >
As shown in FIG. 2A, the Hn-Mc-DR peptide of the present invention showed almost no cytotoxicity, while the Hn-Mc-DRW peptide showed 20% Of hemolysis. On the other hand, melitin, which was used as a positive control, showed high hemolysis of erythrocytes.
2B, the Hn-Mc-DR peptide of the present invention, the Hn-Mc-Del peptide, and the Hn-Mc-DWR peptide showed little cytotoxicity, while the Hn- 29% cell death was observed. On the other hand, melitin, used as a positive control, was highly cytotoxic.
Experimental Example 3 Inhibition of cell membrane-destructive activity and photosynthesis of microalgae induced by red tide of Hn-Mc-DWR peptide
To elucidate the cell membrane-destructive ability of the red tide-induced microalgae of the Hn-Mc-DWR peptide synthesized in Example 1, H. akashowo and pro- After culturing the minimum ( P. minimum ), the cultured microalgae were treated with Hn-Mc-DWR peptide and treated with Sytox-Green fluorescent dye reacting with the intracellular nucleic acid, Respectively.
In order to measure the antifungal activity of the Hn-Mc-DWR peptide and to determine the content of chlorophyll a in the red tide organism, H. akashowo and P. minimum were mixed with F2 And then cultured. Cultured blooming microalgae were inoculated into 12-well microplates at a concentration of 10 5 / ml and cultured at 22 ° C for 1 hour. After incubation, the samples were centrifuged and 90% acetone, an extraction solvent, was added and homogenized. The shredded sample was shaken at -20 ° C to liberate chlorophyll-a and left for 4 hours, then the sample was centrifuged and the supernatant was carefully removed. The amount of chlorophyll-a in the supernatant was then briefly determined using the absorbance of whole cells and the following equation (2). The results are shown in Fig.
As shown in FIG. 3A, microalgae treated with the Hn-Mc-DWR peptide showed remarkable increase in green fluorescence over time.
In addition, as shown in FIG. 3B, it was observed that the amount of chro- phoric acid in the algae treated with the Hn-Mc-DWR peptide decreased significantly over time.
Therefore, it was found that the Hn-Mc-DWR peptide according to the present invention destroys cell membranes, kills harmful algae, and inhibits the growth of microalgae induced by blooms.
Experimental Example 4 Aquarium Biological Experiment of Hn-Mc-DWR Peptide
To verify the anti-algae activity of Hn-Mc-DWR peptide synthesized in Example 1, placed into the scallops in seawater aquarium (length 35 cm × vertical 35 cm ×
As shown in FIG. 4, the negative control treated with only red tide-induced microalgae showed severe damage to the internal organs as compared with the control group not treated with red tide-induced microalgae and Hn-Mc-DWR peptide, Experimental groups treated with the Mc-DWR peptides were observed to have preserved internal organs similar to control groups without red tide-induced microalgae and Hn-Mc-DWR peptides.
Thus, it was confirmed that the Hn-Mc-DWR peptide of the present invention inhibits the proliferation of micro tide microalgae and thus survives marine organisms.
As described above, the Hn-Mc peptide, the Hn-Mc-Del peptide, the Hn-Mc-DRW peptide, and the Hn-Mc-DWR peptide produced by the present invention inhibit the movement and growth of the blooming microalgae, In particular, Hn-Mc-DWR peptides did not show cytotoxicity against fish and shellfish. In particular, Hn-Mc-DWR peptide destroyed the cell membrane of red tide-induced microalgae and decreased the content of chlorophyll a, resulting in the death of red tide-induced microalgae.
Therefore, the Hn-Mc peptide, the Hn-Mc-Del peptide, the Hn-Mc-DRW peptide, and the Hn-Mc-DWR peptide prepared in the present invention can be effectively used as a safe red tanning composition in a marine ecosystem.
<110> Sunchon University Industry-Academic Cooperation Foundation <120> Chimeric peptide having antialgae activity and composition for control of red tide comprising the same <130> PA-15-0166 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Hn-Mc peptide sequences <400> 1 Phe Lys Arg Leu Lys Lys Leu Ile Ser Trp Ile Lys Arg Lys Arg Gln 1 5 10 15 Gln <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Hn-Mc-Del peptide sequences <400> 2 Phe Lys Arg Leu Lys Lys Leu Ile Ser Trp Ile Lys Arg Lys Arg 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Hn-Mc-DRW peptide sequences <400> 3 Phe Lys Arg Leu Lys Lys Leu Ile Arg Trp Ile Lys Arg Lys Trp 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Hn-Mc-DWR peptide sequences <400> 4 Trp Lys Arg Leu Lys Lys Leu Ile Arg Trp Ile Lys Arg Lys Arg 1 5 10 15
Claims (3)
Wherein the chimeric peptide is selected from the group consisting of Alexandrium catanella , Alexandrium tamarense , Chattonella marina , Chattonellar sp. , Cochlodinium polykrikoides , In the group consisting of Gyrodinium impudicum , Hetrosigma akashiwo , Heterosigma sp . Prorosentrum micans , and Prorocentrum minimum , Wherein the chimeric peptide has anti-algae activity against at least one selected red tide-inducing microalgae.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019235682A1 (en) * | 2018-06-07 | 2019-12-12 | 부경대학교 산학협력단 | Novel tetra-peptide and derivative thereof, and anti-red tide or antimicrobial composition comprising same |
| KR20210119754A (en) | 2020-03-25 | 2021-10-06 | 한국생명공학연구원 | Composition for controlling red tide comprising Fictibacillus sp. or their medium as effective component |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107140742A (en) * | 2017-06-05 | 2017-09-08 | 舟山市普陀兴海养殖优质种苗选育研究所 | A kind of mixed type algae water cleanser and its application |
| KR101971250B1 (en) * | 2017-12-19 | 2019-04-22 | 한국생명공학연구원 | Olleya sp. M5A2M strain having algicidal activity against Alexandrium tamarense and Cochlodinium polykrikoides and uses thereof |
| KR102135037B1 (en) * | 2019-05-28 | 2020-07-20 | 한국생명공학연구원 | Erythrobacter sp. 3-20A1M strain producing pyrrole-2-carboxylic acid and having algicidal activity and uses thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019235682A1 (en) * | 2018-06-07 | 2019-12-12 | 부경대학교 산학협력단 | Novel tetra-peptide and derivative thereof, and anti-red tide or antimicrobial composition comprising same |
| KR20210119754A (en) | 2020-03-25 | 2021-10-06 | 한국생명공학연구원 | Composition for controlling red tide comprising Fictibacillus sp. or their medium as effective component |
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